Cellulosic biomass is useful as a raw material for producing various kinds of organic acid compounds, ethanol, or the like by a biological method. The usefulness of cellulosic biomass is attributable to its availability. Unlike sugar-based biomass, such as cone starch and sugar, cellulosic biomass is readily available in various forms, such as agricultural waste or woody waste, at low cost, and therefore is not supposed to be any future obstacle to securing food resources. Cellulosic biomass comprises about 35 to 45% by mass of cellulose, about 30 to 40% by mass of hemicellulose, about 10% by mass of lignin, and about 10% by mass of other ingredients. Cellulose is a polymer of glucose (a hexose). Meanwhile, hemicellulose mainly consists of pentoses, such as xylose and arabinose.
The hemicellulose of cellulosic biomass, such as corn stover, wheat straw, rice straw, and bagasse, mainly consists of D-xylose. Therefore, for effective utilization of cellulosic biomass, it is imperative to establish a technology in which D-xylose is effectively utilized by a biological method.
In a cellulosic biomass utilizing technology, it is necessary to firstly saccharify the raw material into monosaccharides, such as hexose and pentose, and for a reason of process design, these monosaccharides coexist in a culture medium for producing organic compounds. Usually, in such a case, so-called “glucose repression” of a pentose by a hexose occurs. Glucose repression, which makes it impossible to utilize a hexose and a pentose in parallel and simultaneously, is a factor complicating process design and operation control, and is a hindrance to establishing a technology for industrial and effective utilization of biomass. Improvement in this regard is also desired.
Many microorganisms which utilize glucose, a kind of hexose, to produce various kinds of organic compounds by fermentation are well known, and some microorganisms which utilize D-xylose, a kind of pentose, to produce ethanol etc. are also known.
Patent Literature 1 and Non Patent Literature 1 disclose a technology in which wild-type Saccharomyces cerevisiae incapable of utilizing D-xylose is provided with a D-xylose-utilizing ability by transferring 3 genes, that is, a xylose reductase gene and a xylitol dehydrogenase gene both from Pichia stipitis, and a xylulokinase gene from Saccharomyces cerevisiae via plasmids; and a D-xylose-utilizing technology with use of the obtained Saccharomyces cerevisiae transformant. However, the Saccharomyces cerevisiae transformant does not have a sufficient rate of utilizing D-xylose, and in the presence of both D-glucose and D-xylose, glucose repression in D-xylose utilization (in the presence of D-glucose, the rate of D-xylose utilization is extremely slowed) is observed. In other words, an effective D-xylose-utilizing technology has yet to be established. Therefore, improvement regarding these points is desired.
Non Patent Literature 2, also relating to a technology involving Saccharomyces cerevisiae, discloses a technology to isolate a variant having an improved rate of uptake and assimilation of D-xylose, the variant obtained by creating a Saccharomyces cerevisiae transformant that has a disrupted aldose reductase gene and highly expresses a xylulokinase gene, a ribulose 5-phosphate isomerase gene, a ribulose 5-phosphate epimerase gene, a transketolase gene, and a transaldolase gene from Saccharomyces cerevisiae, in addition to a xylose isomerase gene from a fungus, Piromyces sp., and then performing anaerobic xylose-limited chemostat and subsequent anaerobic automated sequencing-batch reactor, and a D-xylose-utilizing technology with use of the obtained strain. In this technology, the rate of D-xylose utilization is improved as compared in the technologies of the above Patent Literature 1 and Non Patent Literature 1 but still insufficient. In addition, in the presence of both D-glucose and D-xylose, glucose repression in D-xylose utilization is still observed. Therefore, improvement regarding these points is desired.
Patent Literature 2 discloses a D-xylose-utilizing technology with use of a Zymomonas mobilis transformant obtained by transferring four genes, that is, a xylose isomerase gene, a xylulokinase gene, a transketolase gene, and a transaldolase gene from Escherichia coli via plasmids into wild-type Zymomonas mobilis incapable of utilizing D-xylose and by allowing the genes to be expressed.
Patent Literature 3 discloses a D-xylose-utilizing technology with use of a Zymomonas mobilis transformant similarly obtained by integrating four genes, that is, a xylose isomerase gene, a xylulokinase gene, a transketolase gene, and a transaldolase gene from Escherichia coli into the chromosome of wild-type Zymomonas mobilis and by allowing the genes to be expressed, the four genes in the transformant being stabler. In these known technologies, simultaneous utilization of hexose and pentose is achieved to some extent, but the D-xylose consumption rate (utilization rate) is insufficient as compared to the D-glucose consumption rate (utilization rate), and therefore, further improvement is needed for establishing a practical technology of simultaneous utilization of hexose and pentose.
Although a wild-type strain of Escherichia coli is capable of utilizing pentoses including D-xylose, it is known that the above-mentioned glucose repression affects D-xylose utilization in the simultaneous presence of D-glucose and D-xylose. Non Patent Literature 3 and Non Patent Literature 4 report that a microorganism capable of utilizing D-glucose and D-xylose simultaneously can be obtained by disrupting the ptsG (glucose phosphotransferase system (PTS) transport) gene responsible for glucose uptake. However, Escherichia coli has a problem of being susceptible to changes in conditions of process operation, resulting in lysis. In addition, the ethanol resistance of Escherichia coli is lower than that of Saccharomyces cerevisia and Zymomonas mobilis. Therefore, ethanol production with use of Escherichia coli has a problem of lower final concentration of ethanol (enormous energy is required for concentration and purification of ethanol from fermentation broth).
Corynebacterium glutamicum and recombinant strains thereof are useful microorganisms for effective utilization of saccharides because they can produce organic compounds, without proliferation of themselves, in bioconversion from saccharides to organic compounds, such as an organic acid, under reducing conditions (Patent Literature 4). In addition, since no reactor volume for proliferation is needed, it is possible to design a compact reactor. The inventors have already disclosed a technology in which a pyruvate decarboxylase gene and an alcohol dehydrogenase gene derived from Zymomonas mobilis are transferred into Corynebacterium glutamicum and expressed for highly effective production of ethanol (Non Patent Literature 5).
In a fermentation process in which a microorganism that proliferates in material production, for example, Saccharomyces cerevisiae, Zymomonas mobilis, or Escherichia coli, is used, a major problem is catabolite repression caused by so-called “fermentation inhibitors” (proliferation inhibitors), such as phenols, furans, and organic acids, produced in a pretreatment step necessary for obtaining, as raw materials, saccharides from cellulosic biomass. It has become clear that a technology involving Corynebacterium glutamicum has an advantage of being free from catabolite repression caused by so-called “fermentation inhibitors” (proliferation inhibitors) for the reason that the technology enables bioconversion to organic compounds, such as organic acids, without proliferation (Non Patent Literature 6).
However, while having an ability of bioconversion with various advantages, a wild-type strain of Corynebacterium glutamicum is originally incapable of utilizing pentoses, such as D-xylose. In this context, the inventors have already disclosed a technology for providing Corynebacterium glutamicum with a D-xylose-utilizing ability by transferring a xylose isomerase gene and a xylulokinase gene derived from Escherichia coli, and allowing them to be expressed (Non Patent Literature 7).
However, even in the recombinant strain of Corynebacterium glutamicum having a D-xylose-utilizing ability, the D-xylose utilization rate is not sufficient as compared with the D-glucose utilization rate. Therefore, further improvement in the D-xylose utilization rate has been required.
Meanwhile, the inventors have proposed a technology of providing Corynebacterium glutamicum with an ability of utilizing L-arabinose, a kind of pentoses contained in a cellulosic biomass raw material (Patent Literature 5).
Specifically, in the technology of Patent Literature 5, an arabinose isomerase gene, a ribulokinase gene, and ribulose-5-phosphate-4-epimerase gene derived from Escherichia coli are transferred into Corynebacterium glutamicum R (FERM P-18976) incapable of utilizing L-arabinose, and allowed to be expressed. Then, into the resultant coryneform bacterium transformant, an L-arabinose transport system proton symporter derived from Corynebacterium glutamicum ATCC31831 is transferred. The newly created transformant has a considerably improved L-arabinose utilization rate and, in the presence of both D-glucose and L-arabinose, can perfectly utilize D-glucose and L-arabinose in a simultaneous manner because glucose repression in L-arabinose utilization is completely canceled.
However, as mentioned above, no corynebacterium transformant in which glucose repression in D-xylose utilization in the presence of both D-glucose and D-xylose is completely canceled and effective simultaneous parallel utilization of D-glucose and D-xylose is achieved has yet been developed.